Fusion protein (RE)

Purpose of this page

This web page is designed to aid the design of a pair of PCR primers that can be used to sub clone a fragment of an open reading frame such that the fragment forms a fusion protein with an open reading frame in the vector.

A Windows program that duplicates this page can be downloaded here.

Copy and paste the VECTOR sequence in to the text box below.

Copy and paste the INSERT sequence in the text box below.

Enter the first primer sequence in the forward direction

Enter the second primer sequence in the forward direction

Instructions

Step 1

Copy and paste your vector and insert into the text boxes on the first page (use the reverse button to reverse complement the sequence). Next enter the sequence of the forward and reverse primers you wish to use. Finally press the 'Submit' button.

Step 2

Select the restriction enzymes you wish to use in the vector and the position of primers in the inserts 9ideally there shoulsd be only one site). While the enzyme lists state 5' and 3' the enzymes will be automatically sorted by position. Next press 'Submit' button and view the picture showing the open reading frames in the construct. To adjust the junction between the vector and insert open reading frames change the values in the 5' and 3' junction list boxes and press the 'Submit' button again. This changes the position of the primer in the insert sequence while maintaining the primer length.

The sequence of the primer oligonucleotides (plus restiction sites0 is shown in the lower text box along with the sequence of the resultant construct. Since some enzymes need DNA flanking the restriction site to efficiently cut the DNA four extra base are added to the 5' end of the primers. These are lost during the cloning.